Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice

doi:10.12122/j.issn.1673-4254.2024.08.20

Abstract

Abstract:

Objective To investigate the effects of platelet-specific Rictor knockout on platelet activation and thrombus formation in mice. Methods PF4-Cre and Rictor^fl/fl transgenic mice were crossed to obtain platelet-specific Rictor knockout (Rictor-KO) mice and wild-type mice (n=65), whose expression levels of Rictor, protein kinase B (AKT) and p-AKT were detected using Western blotting. Platelet counts of the mice were determined using routine blood tests, and hemostatic function was assessed by tail vein hemorrhage test. Venous thrombosis models were established in the mice to evaluate the effect of Rictor knockout on thrombosis. Platelet aggregation induced by ADP and thrombin was observed in Rictor-KO and wild-type mice, and flow cytometry was used to analyze the expression levels of integrin αIIbβ3 and CD62P in resting and activated platelets. Plasma PF4 levels were determined with ELISA. Megakaryocytes from Rictor-KO and wild-type mice wereincubated by vWF immunohistochemical antibody and APC-CD41 antibody to detect the number and ploidy of megakaryocytes, respectively. Platelet elongation on collagen surface was observed with scanning electron microscopy. Results Compared with the wild-type mice, Rictor-KO mice showed significantly decreased AKT phosphorylation, decreased platelet production, reduced thrombosis, and decreased platelet activation in response to ADP and thrombin stimulation. The Rictor-KO mice also showed lowered expression level of P-selectin protein and activation of integrin αIIbβ3 with suppression of platelet extension, reduced plasma PF4 level and decreased number of megakaryocytes in the bone marrow. The ploidy of megakaryocytes and the mean area of proplatelets were both significantly decreased in Rictor-KO mice. Conclusion Platelet-specific Rictor knockout inhibits platelet generation and activation to result in decreased thrombus formation in mice, suggesting the potential of mTORC2 activity inhibition as an efficient antithrombotic strategy.

Key words: Rictor, platelets, thrombosis

Qing LONG, Jun YANG, Anling LIU. Platelet-specific Rictor knockout inhibits platelet production and activation and reduces thrombosis in mice[J]. Journal of Southern Medical University, 2024, 44(8): 1605-1611.

Figures/Tables 4

Fig.1 Generation of platelet-specific Rictor knockout (KO) mouse models. A: Genotyping results. B: Immunoblotting for Rictor, Akt, and p-Akt (S473) in the platelets. C, D: Gray scale of Rictor (C) and p-Akt (D) measured by Image J. E: Platelet counts of control and KO mice. *P<0.05,***P<0.001.

Fig.2 Rictor deletion reduces susceptibility to thrombosis in mice. A: Average tail bleeding time of Con and KO mice (n=10). B-D: Images (B), lengths (C), and weights (D) of thrombus from Con and KO mice. *P<0.05.

Fig3 PF4-Rictor mice exhibit severe deficiency in platelet function. A, B: Platelet aggregation induced by ADP (A) and thrombin (B) in PRP. C-D: FACS analysis of αIIbβ3 (C) and CD62P (D) in resting platelets. E, F: FACS analysis of αIIbβ3 (E) and CD62P (F) in platelets pretreated with thrombin. G: Platelet spreading on fibrinogen. H: ELISA analysis of plasma PF4 levels. *P<0.05, **P<0.01.

Fig.4 Rictor deletion inhibits megakaryocyte maturation. A: vWF staining of femoral bone sections from Con and Rictor-KO mice (Scale bar=200 μm). B: Ploidy formation of 3-month-old Con and KO mice. C: Ultrastructure and proplatelet formation of megakaryocytes from Con and KO mice (Scale bar=2 μm). *P<0.05, **P<0.01.

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